Studies of Co·Bleomycin A2 Green:  Its Detailed Structural Characterization by NMR and Molecular Modeling and Its Sequence-Specific Interaction with DNA Oligonucleotides

Abstract

The structure of homogeneous Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) has been determined using 2D NMR methods and molecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907−916) reported several possible structures for CoBLM A2 green compatible with their NMR data acquired on a mixture of CoBLM A2 green and A2 brown forms. The availability of the pure CoBLM A2 green, which is stable for months at neutral pH, has allowed the complete assignments of the 1H and 13C chemical shifts, observation of 55 intramolecular NOEs, and determination of 15 coupling constants allowing the definition of dihedral angles. These results are a prerequisite to determining its structure with duplex DNA of a defined sequence (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). Two screw sense isomers each containing two possible axial ligands (the primary amine of the β-aminoalanine and the carbamoyl nitrogen of the mannose) were considered as viable candidates for the structure of CoBLM A2 green. Using the NMR constraints and molecular dynamics calculations, the structures of all four isomers were generated. One set of screw sense isomers can be readily eliminated from considerations based on violations of NOE and dihedral angle constraints. The other screw sense isomer containing either one or the other of the postulated axial ligands has been examined in some detail. The structure containing the primary amine of β-aminoalanine as the axial ligand is favored on the basis of coupling constants and NOE arguments, potential energy considerations, model studies, and studies with analogs of BLM. The favored structure is compact with the bithiazole moiety folded back underneath the equatorial plane of the metal binding domain, on the same face as the hydroperoxide ligand. The geometry of the peptide linker is very well defined by the observed coupling constants in the valeryl and threonine moieties of the linker. CoBLM A2 green has been studied with two self-complementary oligonucleotides, d(CCAGGCCTGG) and d(CCAGTACTGG). Both of these oligomers possess a single, UV light-mediated cleavage site (C and T, respectively). In addition, fluorescent quenching studies have allowed the determination of the first sequence-specific dissociation constants of 1.7 × 10-7 and 1.5 × 10-7 M, respectively. Titration of CoBLM A2 green with each of these oligomers reveals a 1:1 complex in slow exchange on the NMR time scale. The upfield shifts of the bithiazole protons in both of these complexes are indicative of a partial intercalative mode of binding. The stage is now set for the determination of the structure of the CoBLM A2 green bound sequence specifically to DNA.

Crystal Structures of the Transposon Tn5-carried Bleomycin Resistance Determinant Uncomplexed and Complexed with Bleomycin

“…Furthermore, the primary amine of the ␤-aminoalanine is just positioned over the putative equatorial plane for the metal ion with the same chirality as the propositions (45)(46)(47). Judging from these observations, the primary amine of the ␤-aminoalanine moiety may be an axial ligand.…”

“…Most investigators agree on the fact that the equatorial ligands are the secondary amine of the ␤-aminoalanine moiety, the amide nitrogen of the ␤-hydroxyhistidine moiety, and the nitrogens from the pyrimidine and the imidazole rings. However, three possibilities are emphasized for the axial ligand: (i) the primary amine of the ␤-aminoalanine moiety (45)(46)(47); (ii) the carbamoyl nitrogen of the mannose moiety; and (iii) the primary amine of the ␤-aminoalanine moiety and the carbamoyl nitrogen of the mannose moiety (48 -50). Although our model of BLMT complexed with Bm does not contain the metal ion, we suggest that the primary amine of the ␤-aminoalanine moiety is suitable as an axial ligand for the metal ion.…”

Sequence-specific Changes in the Metal Site of Ferric Bleomycin Induced by the Binding of DNA

“…Manderville et al (44,45) In their NMR study of DNA-bound HOO-Co(III)-BLM, Wu et al (19,20) indicate that the 4-NH 2 group and N-3 of the pyrimidine ring of the drug hydrogen bond with N-3 and the 2-amino group, respectively, of the guanine residue in the drug's cleavage site. Based on our EPR investigation of Fe(III)-BLM, the reduction in the number of conformers that the iron drug complex can achieve with d(CGCGCG) or DNA, but not with d(ATATAT) (Fig.…”

Non-heme iron enzymes: Contrasts to heme catalysis

“…H-atom abstraction will be still more favorable than homolysis because of the concerted formation of the HOO bond along the reaction coordinate. From frontier molecular orbital theory, this HOO bond formation requires proper substrate orientation, which seems to be achieved in the ABLM͞DNA complex (36). Thus, compared with heme systems, the heterolytic cleavage of the OOO bond in a non-heme environment is energetically much less favorable, due, in part, to the difficulty in oxidizing the non-heme iron ligand.…”